Metal ions play important roles in many biological systems. Cells utilize metal ions for a wide variety of functions, such as regulating enzyme activities, protein structures, cellular signaling, as catalysts, as templates for polymer formation and as regulatory elements for gene transcription. Metal ions can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete. A large number of natural and synthetic materials are known to selectively or non-selectively bind to or chelate metal ions. Ion chelators are commonly used in solution for in vivo control of ionic concentrations and detoxification of excess metals, and as in vitro buffers. Ion chelators can be used as optical indicators of ions when bound to a fluorophore, and may be useful in the analysis of cellular microenvironments or dynamic properties of proteins, membranes and nucleic acids. For example, Ca2+ ions play an important role in many biological events, and so the determination of intracellular Ca2+ is an important biological application.
Fluorescent indicators utilizing a BAPTA chelator have been predominantly used for intracellular calcium detections (see U.S. Pat. No. 4,603,209; U.S. Pat. No. 5,049,673; U.S. Pat. No. 4,849,362; U.S. Pat. No. 5,453,517; U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; U.S. Pat. No. 5,501,980; U.S. Pat. No. 5,459,276; and U.S. Pat. No. 5,516,911; each of which is hereby incorporated by reference). Fluorescein-based fluorescent calcium indicators (such as Fluo-3 and Fluo-4) are the most common fluorescent indicators used in biological assays. However, these existing xanthene-based calcium indicators typically have either small cell-induced excitation and fluorescence wavelength change, and/or low calcium-induced fluorescence enhancement, resulting in low detection sensitivity and high assay background. In addition, the existing xanthene-based calcium indicators have short emission wavelength, which often interferes with the fluorescence of some agonists and/or antagonists to be screened. Another drawback is resulted from the spontaneous hydrolysis of monoalkylated fluorescein-based indicators in cell medium, generating significant assay background.
In view of the existing drawbacks for currently used fluorescein-based fluorescent calcium indicators, what is needed are improved compositions and methods that offer sensitive detection of small variations in calcium and other ion concentrations, with a rapid response and a strong fluorescence signal. Also needed are fluorescent indicators that can be readily loaded into live cells with large spectral shift and fluorescence enhancement. Another preferable property is that the indicators have emission at longer wavelength to reduce the interference from the fluorescence of agonists and antagonist that often have fluorescence at short wavelength. In addition, compositions and methods that are less susceptible to the effects of external changes (such as temperature) are preferred for high throughput screening and high content analysis.
The present application is directed to a family of fluorescent dyes that are useful for preparing fluorescent metal ion indicators. The indicators include a carbofluorescein lactone fluorophore that is incorporated with an ionophore, and are useful for the detection, discrimination and quantification of metal cations. The fluorescent indicators of this invention demonstrate unexpected larger spectral shift upon cell-induced hydrolysis, unexpected emission shift to the longer wavelength, better stability in the presence of cells and better cellular retention compared to the existing fluorescein ion indicators.